PROJECT SUMMARY/ABSTRACT The major obstacle to the successful application of human cardiac stem cell biology is the immaturity of in vitro stem cell-derived cardiomyocytes. Genetic manipulations of stem cell-derived cardiomyocytes have not been successful in achieving the maturity sufficient for regenerative medicine, drug screening, disease modeling and developmental biology. Recent multi-center study revealed the importance of non-genetic contributors to the development of congenital heart disease. Thus, in both in vitro and in vivo settings, non-genetic factors are understudied area of research that can, combined together with the wealth of knowledge in genetic contributors to cardiogenesis, potentially solve the immaturity issue of stem cell-derived cardiomyocytes. In fact, the metabolic/nutritional environment is a major non-genetic factor that impact heart formation. It is well-established that maternal hyperglycemia is associated with significant increase in the risk of congenital heart disease. However, little is known about whether high glucose directly impact the differentiation of cardiomyocytes and how high glucose might impact the flow of downstream metabolic pathways. Glucose is the most critical nutrients to the cells and its metabolism is tightly regulated in any cells. In the fetal heart, glucose is taken up through transporter isoforms 1 and 4 and processed through multiple catabolic and anabolic pathways including glycolysis, TCA, pentose phosphate pathway, hexosamine pathway, etc. Our preliminary data with human embryonic stem cell-derived cardiomyocytes and murine model of diabetic pregnancy suggest that it is not the catabolic extraction of energy but the anabolic biosynthesis of nucleotides from glucose that plays a major role in regulating cardiogenesis during fetal stage. These results have led to our central hypothesis that glucose inhibits fetal cardiac maturation via nucleotide biosynthesis. This proposal will test it by genetic, metabolic, and physiological analyses in vivo and in vitro. The results are expected to demonstrate that unique metabolic environment of fetal heart is not merely a consequence of genetic differentiation program but also a driver of cardiac maturation. By focusing on understudied area of cardiogenesis research, this study will add another dimension to our understanding of cardiogenesis and congenital heart disease.